Cells in the ciliary epithelium (CE) of the eye can clonally expand in culture to produce spheres of cells that differentiate into retinal neurons and glia. It is believed that these spheres are produced by retinal stem cells (RSCs) and hold promise for cell-based therapies to treat degenerative retinopathies. To improve our understanding of RSC expansion and differentiation, we have carried out a series of preliminary studies on human and mouse CE-derived spheres. Our data have led us to reconsider the current retinal stem cell model and to propose a new hypothesis on the expansion and differentiation of CE-derived cells. We propose that the pigmented CE cells rather than RSCs, expand to form spheres and subsequently transdifferentiate into rods, bipolar cells and M[unreadable]ller glia. This proposal will test if CE-derived spheres form by proliferative expansion of pigmented CE cells or by an RSC mechanism (Aim 1). Culture experiments and in vivo transplantation studies will also be done to test whether pigmented CE cells transdifferentiate into retinal neurons and glia, or if those cells are produced from retinal stem/progenitor cells (Aim 2). Distinction between these 2 hypotheses must be made, because the pathways regulating proliferation and differentiation in retinal stem/progenitor cells differ from those in epithelial cells, and efforts to optimize expansion and differentiation of CE-derived cells must focus on the pathway that reflects that system's biology. The successful completion of these experiments may substantially affect future development of cell-based therapies for millions of people worldwide who suffer from retinal degeneration. This research proposal will also move the RSC field forward by resolving several outstanding questions regarding the proliferation and differentiation of CE-derived cells in culture and in vivo. PUBLIC HEALTH RELEVANCE: Retinal degeneration affects millions of people worldwide each year. One approach that is being considered to treat degenerative retinopathies is the use of retinal stem cell-based therapy to repopulate the photoreceptors and other cells that are lost. Retinal degeneration is an ideal candidate for such cell-based therapy because the eye is easily accessible for therapeutic intervention and the immunoprivileged status of the eye makes rejection of grafted cells less likely than in other organs where stem cell-based therapies are being considered. However, one of the limitations of the retinal stem cell field compared to other areas of stem cell biology is that it is a relatively new area of research. Cells with properties of stem cells in the ciliary epithelium of the eye were first discovered just 7 years ago. As a result, there are still some important questions that must be answered before we can move forward with therapeutic intervention in humans using retinal stem cells. Specifically, we need to better understand the mechanism of retinal stem cell growth and differentiation in culture. The successful completion of the experiments proposed here will resolve whether there is a retinal stem cell in the ciliary epithelium of the mammalian eye or if pigmented ciliary epithelial cells simply expand and transdifferentiate into retinal neurons and glia. These data are essential for directing future studies on cell-based therapies for retinal degeneration and will have a major impact on moving the retinal stem cell field forward. [unreadable] [unreadable] [unreadable]